A Molecular Orbital Study of the Chloramphenicol Family of Drugs: A

Preliminary Report

RICHARD E. BROWN, ALFRED0 M. SIMAS, AND ROY E. BRUNS

Instituto de Qujmica, Unioersidade Estadual de Campinas, Campinas, S.P.. B r a d

Abstract

CNDO/2 calculations were completed on various cogeners of the chloramphenicol molecule to study the potency of this antibiotic. Several MO indices correlated well with the biological activities. Little effect upon the stabilization of the radical intermediate was found. The substituents apparently alter significantly only the electrostatic interactions with the receptor.

1. Introduction

One of the most effective antibiotics is the chloramphenicol molecule which has proven to be useful against some diseases, in particular typhoid fever. Many cogeners of this molecule have been synthesized in an attempt to find a drug of similar biological activity ( BA) but reduced toxicity. Only one derivative has yielded a higher activity. Since the drug is relatively easy to modify and test, considerable data are available on its structure-activity relationships (SAR) [ I -31. Experimental and theoretical studies have also been made to determine the conformation of the drug [4,5].

Three segments of the drug are critical to its activity, while substitutions have been made at two points on this molecule without destroying the biological ac- tivity. The propanediol moiety cannot be altered in any manner. The other two segments, the acylamino side chain and thep-nitro phenyl group are critical to the activity, but can be modified while retaining the biological activity. Ac- ceptable modifications include the replacement of the dichloromethyl group in the acylamino moiety (R series) and the replacement of the p-nitro group on the benzene ring with other meta or para groups (X series). Nearly 40 published cogeners of this drug have been prepared and tested. Only the replacement of the dichloromethyl group by a trifluoromethyl group gives a more active drug.

Our interest in these molecules is threefold. We wish to elucidate the mech-

InIernalional Journal of Quantum Chemistry: Quantum Biology Symposium 4,357-362 (1977) Q 1977 by John Wiley & Sons, Inc. 3 5 1

358 BROWN, SIMAS, AND BRUNS

anism of the chloramphenicol potency. Conflicting theories still exist on the critical structural features of this molecule which determine the biological ac- tivity and the functions which the substituents play in this. Second, we wish to test the applicability of some molecular orbital indices in explaining and cor- relating biological activities. Finally, we eventually want to apply these indices to the prediction of the activity of yet unstudied cogeners of the chloramphenicol drug.

2. Method Various molecular orbital ( MO) indices have been demonstrated to effectively

correlate with the molecular reactivities of the aromatic hydrocarbons, the substituted benzoic acids, and the substituted phenylamines [ 6 ] . These indices were calculated with the C N D 0 / 2 (complete neglect of differential overlap) method, which apparently reproduces the necessary trends to parametrize the reactivity of these compounds via a regression analysis. Acceptable correlations were obtained for both the ring and exocyclic sites of reactivity. More studies on these indices are currently being made, for example, the separation into u and K components, but we feel the initial results warrant sufficient confidence for an application to the SAR of the chloramphenicols.

The MO indices of interest to us are the net atomic charge at atom k, Q k ; the electrophilic net group charge Qwx..., for the atoms W X 2; the electrophilic and nucleophilic MO densities at atom k, qE,k , q N , k ; the MO energies €- i s f + i ;

the atom-atom polarizabilities 7rk.J; the nucleophilic and electrophilic super- delocalizabilities S N , k and S E , k ; and the electric field and potential energy for a unit positive charge at point R near the molecule, E ( R ) and V(R) . In this notation k and 1 refer to atoms while i a n d j refer to a particular MO. Although not implemented here, the indices for the ring atoms should be partitioned into their u and K components. In our notation the MO energies decrease in order ..- c+2, € + I , E - I , E - Z , .a. such that the energy of the highest occupied MO (HOMO) is c-1 and that of the lowest unoccupied MO (LUMO) is c + ~ . The frontier variants of some of these indices are denoted with respect to the orbital, for example, S - l , k , S + 2 , k , q + 2 , k . etc., where the indices with the signs refer to the relevant MO. The frontier polarizabilities are calculated with both the HOMO and LUMO and are denoted as ?r'k,k and K'k,J, respectively. These indices have found wide applicability with the Hiickel method and except for the superdelocalizabilities and polarizabilities, have been successfully applied with the C N D 0 / 2 method to study chemical reactivity [7-91. Wohl applied all these indices with the ex- tended Huckel method (EH) to the problem of drug design for the benzothia- diazine-like-antihypertensive agents [lo]. A complete explanation of these in- dices, adequately translated to the more advanced C N D 0 / 2 method, is given in [6] and will not be repeated here.

Linear regressions will be made in an attempt to parametrize the known bi- ological activities (BA) of the drugs in terms of these indices x,.

BA = (Am f 6A,)x, + B f 6B m

MO STUDY OF CHLORAMPHENICOL 3 5 9

We should point out that all the indices need not be MO indices as discussed above. We can add others to this set to include nonelectronic properties, such as a parameter to measure the steric effect of the substituents which these MO properties will not monitor effectively. The molar refractivities or the van der Waals contact distances could be used in this capacity [ 1 1, 121. In addition other experimental parameters, such as the hydrophobicity, can be used to monitor the effect of the interaction of the drug with the aqueous environment [ 131.

3. Results and Discussion

We report here some tentative results. Only single variable regressions have thus far been completed using the MO indices discussed above. Calculations on additional cogeners are in progress, and the results at the multiple variable level will be reported later. However, these results do illustrate some interesting points about the chloramphenicol potency. In all these calculations we use the most stable conformation as determined experimentally by Bustard, Egan, and Perun [4] and as confirmed theoretically by Holtje and Kier [5]. (Conformation I is described in [ 5 ] . )

Eight cogeners have been analyzed with X = NO2 and R = CF3, CHC12, CH2C1, CHF2, CH2F, CC13, and CH2CN. This R series showed poor correlation at the single variable level, with the best regressors explaining only about 40% of the variance. Hansch and coworkers found somewhat similar results which explained about 30% of the variance with their two best regressors, the experi- mental polarizability and the Hammett function cr*. They also felt that electron withdrawal is the main function of the R group. However, overly electronegative groups could change the most stable conformation. By arguing that the electron withdrawal functioned by increasing the acidity of the amide nitrogen proton, they played down the role of the carbonyl group. Our tentative results indicate that the indices associated with the carbonyl group correlate better with the BA than do those associated with the N H group. The best regressors are presented in Table 1. The reaction apparently is not frontier controlled since all these indices gave inferior results. The electrostatic indices gave the best results and indicate that the potential generated by the carbonyl group may be instrumental in ini- tiating the reaction associated with the side chain. No definite conclusions can yet be drawn until more analyses are made with more than one variable, espe- cially the steric parameters. However, the poor results obtained with the frontier parameters support the electrostatic nature of the drug-receptor interaction. The effect on the protonation of the amide nitrogen hydrogen, if real, may be of only secondary importance and participate in a non-rate-determining step.

We will discuss in more detail the effect of the para benzene substituents on the activity and those parameters which correlate best. Assuming little steric hindrance of the X group, as these results indicate, a single variable analysis can shed some light on the controversy surrounding the effect of the p substituents. Hansch and coworkers have argued that the p substituents function primarily to stabilize a free radical at the benzyl carbon [3]. Holtje and Kier, however, produced some evidence that this argument was not valid and offered an alter-

360 BROWN, SIMAS, AND BRUNS

TABLE 1. The best single variable regressions with X = NO2 and R = CF3. CHC12, CHzCI, CHF2, CHzF, CCI3, and CHzCN.

Ba - Rb - R2 INDEX .” - Qco QO

QC

QH

-45.5 1.04 .634 .402

-19.0 -3.58 .631 .399

0.691 -3.17 .626 .392

-15.8 6.60 .604 .365

1.88 1.00 .584 .341

7.50 -8.86 .565 .319

‘N,O

QC (R)

C(R) denotes the central carbon atom of the R group; C and 0 denote the carbonyl atoms; H denotes the hydrogen atom in the N H group.

The parameters in the fit, BA = Ax + B. R is the correlation coefficient of the regression.

native mechanism [ 5 ] . By mimicking the chloramphenicol cogeners with smaller model molecules, they showed how neither the atomic charges nor the energy change associated with the formation of the radical correlated with the activities. They proposed instead that the substituted phenyl ring is instrumental in the binding to the receptor and that the p substituents function to modify this in- teraction energy. By modeling the receptor site after a tryptophan side chain and calculating the interaction energies between the p-substituted phenyl ring and the receptor model via the monopole bond-polarizability method of Claverie and Rein [ 5 ] , they obtained a very good correlation with the BA. Their high correlation coefficient of 0.914 with 12 drugs used in the regression was indeed impressive.

For this sequence of cogeners we have completed calculations with R = CHC12 of the parent molecule, and X = NO2, CN, OCH3, C1, H, and NH2. Tables I1 and 111 give some results for this series which illustrate some interesting points. Unfortunately Holtje and Kier did not include the atomic charges of the phenyl group such that we could make comparisons with their model results. They did include the atomic charges on the acylamino moiety for the other set of cogeners where R was varied. For this series our results showed somewhat better trends than their model results, especially for the amide nitrogen. It was at this atom where the rest of the chloramphenicol was replaced by a simple hydrocarbon to make their CNDO calculations tractable. For their model for the substituted phenyl series where X was varied, the entire side chain attached to the benzyl carbon was replaced by a simple hydrocarbon, thus making their results for the benzyl carbon suspect. It would be difficult to illuminate the role of the benzyl carbon on these data alone. They did state that the charges on the hydroxy- methylene moiety remained virtually constant with variation in thep substitu- ents. Our complete CNDO calculations gave the same qualitative results with variations in the atomic charges on this moiety being normally an order of magnitude smaller than those observed on the acylamino moiety for the R series discussed above. These fluctuations are too small to be chemically or biologically significant. Table I1 illustrates this point. The group charge for the hydroxy- methylene moiety Q H ~ H shows a high correlation with the BA, but a coefficient

MO STUDY OF CHLORAMPHENICOL 36 1

TABLE 11. Some single variable regressions with R = CHClz and X = NOz. CN, OCH,, CI, H, and NH2.

- INDEX - Aa - Ba - Rb - R2

QC1 2.53 0.85 .497 .297

QC 2 7.99 1.21 .603 .364

Qc3 -23.4 1.24 .612 .375

Qc4 17.2 1.15 .789 .623

Qc5 -157. 24.7 .723 .523

QO 255. 67.2 .918 .843

Q+ QX+

QHOCH 99.7 2.29 .949 .go1

'N,C5 1.26 -14.2 .946 .a94

'N,O 2.55 -12.4 .941 .885

Qx -6.93 0.594 .956 .913

7.76 0.615 .908 .a24

-26.9 0.843 .831 .690

'N,C4 .177 -1.38 .915 .837

The phenyl carbons are numbered clockwise from the para position with the benzyl carbon numbered as C5 .0 denotes the hydroxyl oxygen attached to the benzyl carbon and 6 the phenyl group.

a Parameters used in the regression analysis, BA = Ax + B. R is the correlation coefficient of the regression analysis.

of nearly 100 in the regression equation. This is large compared to that of the best charge regressor and clearly demonstrates how the variation in the charge is too small to explain the BA. This holds true also for the other indices associated with the hydroxymethylene moiety, including the superdelocalizabilities given in Table 11, which we feel suffer fluctuations that are too small to explain the BA .

Oneinteresting point is how the group charges Qx and Q+ of the p substituent

TABLE 111. Some values of the best regressors for the phenyl substituents versus the biological activity (BA).

V Qx 3 - EZ - X BA - - -

2.00 -. 204 .167 4.22 1.86

CN i.ag -.om .065 2.09 1.09

OCH3 1.21 -.094 .099 1.24 0.70

c1 1.05 -.063 .052 1.29 0.76

H 0.80 -. 005 -. 004 0.74 0.60

NH2 0.50 -.024 .029 0.01 0.47

NO2

The R group here is CHC12. EL is the electric field generated by the molecule perpendicular to the benzene ring and at a point 4.5 A above the center of the ring. V is the Coulombic field at this point.

362 BROWN, SIMAS, AND BRUNS

and the benzene ring correlate very well with the BA while the atomic charges give very poor results. Not surprisingly, the total charge of the substituted phenyl moiety does poorly. All this illustrates how group charges should be studied since they may mimic the Coulombic potential and field produced by the molecule, while the atomic charges may fare poorly in this respect. Table I11 illustrates this point. Clearly the arguments of Holtje and Kier have some merit, and ap- parently the potential produced on this end of the drug explains the BA suffi- ciently. Steric effects are apparently not important here. The loss of the H atom radical must not be involved in a rate-determining step. The superdelocalizability of the benzyl carbon correlates well with the BA, but is of marginal significance, since for this series it varies by only about 2% in magnitude while Qx and Qb showed fluctuations of about 100%. The frontier indices also performed poorly. All this indicates that this reaction is electrostatically controlled where the po- tential generated by the phenyl moiety is intimately involved in the receptor-drug interaction as the initiating step. We feel that the stabilization of the radical plays a secondary role.

Nofe added in proofi Recently Garrett [ 151 has published a modified value for the biological activity of the R = CF3 cogener of chloramphenicol. It’s activity is now reported to be smaller than the one found for the natural drug. This change will affect the values in Table I1 somewhat, although it is extremely unlikely that single variable regressions using the revised value will satisfactorily explain the biological activities. New single variable and multiple variable regression analyses are now being carried out. Their results will be reported in the near future.

Acknowledgment One of the authors (A.M.S.) is indebted to FAPESP, the Fundacao de Am-

paro A Pesquisa do Estado de Sgo Paulo, for the grant of a graduate fellowship. The cooperation and the generous grant of computer time from the Center of Computation of UNICAMP is also greatly appreciated.

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Received February 17, 1977

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